Skin is a versatile organ that serves as a self-renewing and self-repairing interface between the body of a vertebrate organism and its environment. The skin covers the entire external surface of the body. In humans this includes the external auditory meatus, the lateral aspect of the tympanic membrane, and the vestibule of the nose. The skin is continuous with, but distinct from, the mucosae of the alimentary, respiratory, and urogenital tracts; the specialized skin of the mucocutaneous junctions connects the skin and the mucosae. In addition to its protective functions, skin is capable of absorption, and excretion, and is also an important primary site of immunosurveillance against the entry of antigens and initiation of the primary immune response. Skin also performs many biochemical synthetic processes that have both local and systemic effects, and in this sense can be regarded as an endocrine organ. For example, skin is responsible for the formation of vitamin D, and also synthesizes cytokines and growth factors. For a detailed review of skin and its functions, see Gray's Anatomy: The Anatomical Basis of Medicine and Surgery, Williams et al. (eds.) 1995 Churchill Livingstone, New York, pgs. 376-417.
Skin can be divided into two major classes: thin, hairy (hirsute) skin (which covers most of the body), and thick, hairless (glabrous) skin (which forms the surfaces of the palms of the hands, soles of the fee, and flexor surfaces of the digits). Both classes of skin are composed of three basic layers: the epidermis, the dermis, and the hypodermis. The primary differences in the two classes of skin are in thickness of their epidermal and dermal components, and in the presence of hairs with their attendant sebaceous glands and arrector pili muscles (pilasebaceous units).
The epidermis, a stratified keratinous squamous epithelium primarily composed of keratinocytes, can be further divided into several strata (from deep to superficial): stratum basale, stratum spinosum, stratum granulosum, stratum lucidum (where present), and stratum corneum. Epidermal appendages (e.g., pilosebaceous units, sudoriferous gland, and nails) are formed by ingrowth or other modification of the general epidermis, often referred to as the interfollicular epidermis. In addition to keratinocytes, the mature epidermis also contains nonkeratinocytes including melanocytes (pigment-forming cells), Langerhans cells (which are immunocompetent antigen-presenting cells derived form bone marrow), and lymphocytes. The epidermis also include Merkel cells, which are thought to be modified keratinocytes.
The population of keratinocytes undergoes continuous renewal, with a mitotic layer of cells at the base of the epidermis replacing those shed at the surface. In order to maintain a constant thickness, the rate of cell production must equal the rate of cell loss. Thus at any one time in the basal layer of the epidermis there are a variety of keratinocytes in different states of differentiation. These keratinocytes can be classified into three types according to their clonal proliferative capacity: 1) stem cells, which have extensive growth capacity; 2) differentiated paraclones, which have limited growth capacity; and 3) intermediate meroclones, which are thought to constitute long-lived progenitor cells (Trainer et al. 1997 Hum. Mol. Genet. 6:1761-7; Barrandon et al. 1987 Proc. Natl. Acad. Sci. USA 84:2302-6).
The cellular system responsible for both local and systemic humoral and cellular immune responses mediated in the skin is referred to as the skin-associated lymphoid tissue (SALT). SALT primarily comprises Langerhans cells, T cells, and keratinocytes of the epidermis, as well as fibroblasts, macrophages, mast cells, eosinophils, neutrophils, Langerhans cells, T-cells and B cells (including plasmacytes) of the dermis. The Langerhans cell, a key SALT element, belongs to the general group of dendritic cells (DC), mononuclear phagocytic cells important in immunological reactions. Langerhans cells carry receptors for the Fc portion of IgG and for complement components (C3b-C4b and C4d), and express a variety of antigens, including MHC Class I and Class II. Langerhans cells internalize and process antigen, and migrate to the draining lymph nodes to present the antigen to T cells, resulting in T cell activation and proliferation, and generation of cytotoxic T-cells. The keratinocyte, which expresses Ia antigen, produces cytokines that can enhance or downregulate T-cell activation.
The skin, particularly the epidermis, is appealing as a target tissue for delivery of polynucleotides. First, as indicated above, skin mediates a variety of important local and systemic functions, including development of immunity, and expression of polypeptides for local and systemic delivery. These normal skin cell functions can be exploited to mediate expression of a desired recombinant polynucleotide, and to elicit the desired immunological and/or physiological phenomenon. For example, skin has been successfully used as a site for genetic immunization (i.e., immunization by administration of an antigen-encoding sequence) (see, e.g., U.S. Pat. No. 5,589,466; Robinson et al. 1997 Sem Immunol 9:217-83; Johnston et al. 1994 Meth Cell Biol 43:353-65; Barry et al. 1997 Vaccine 15:788-91; Sundaram et al. 1996 Nucl. Acids Res. 24:1375-7; Moelling 1997 Cytokines Cell Mol. Ther. 3:127-35). Second, skin is an attractive target organ due to its accessibility, thereby providing one of the easiest routes of administration. Moreover, because it is a stratified epithelium, skin allows for the possibility of targeting gene expression to either differentiated or proliferative cells, depending upon the desired effect of gene product expression. In addition, epidermal biology is relatively well-characterized at both the cellular and molecular levels. For example, the regulatory sequences of the keratins have been used to express a variety of exogenous genes in the epidermis of transgenic mice, and are readily adaptable for expression in other organisms (Greenhalgh et al. 1994 J. Invest. Dermatol. 103:63S-69S; Vassar et al. 1991 Genes Dev 5:714-27; Bailleul et al. 1990 Cell 62:697-708).
Expression of a recombinant gene product in skin could also be used in the therapy of a wide variety of diseases including acquired or genetic diseases of the epidermis, as well as conditions amenable to treatment by delivery of gene products systemically. Since the epidermis is know to secrete a variety of cytokines (Luger 1990 J. Invest. Dermatol. 95: 100S-104S) and growth factors (Pittelkow et al. 1988 Am NY Acad Sci 548:211-24), the skin could be exploited as a bioreactor designed for the secretion of gene products that have a local or a systemic effect (e.g., factor IX, hGH, etc). For example, the skin could be used as a site of metabolic waste disposal for circulating toxins such as oxyadenosine, the toxin of ADA deficiency (Blaese 1992 Pediatric Res 33(suppl):S49-S55; Flowers et al. 1990 Proc Natl Acad Sci. USE 87:2349-53). For reviews on the use of skin as a target for gene product delivery, see Trainer et al. 1997 Hum Mol Genet 6:1761-7; Greenhalgh et al. 1994 J. Invest Dermatol 103:63S-69S; Khavari et al. 1997 Dermatol Clin 15:27-35).
Conventional methods for delivery of polynucleotides for expression in the skin include invasive, semi-invasive, or non-invasive methods. Invasive methods involve breaking the skin or otherwise disrupting or bypassing the epidermal barrier. Conventional invasive methods include needle injection (U.S. Pat. No. 5,589,466; Masayuki et al. 1996 FEMS Immunol Med Microbiol 14:221-30; Ciernik et al. 1996 Hum Gene Ther 7:893-9), particle bombardment ("gene gun;" Vahlsing et al. 1994 J. Immunol Meth 175:11-22; Cheng et al. 1993 Proc. Natl. Acad. Sci. USA 90:4455-9; Sundaram et al. 1996 Nucl. Acids Res. 24:1375-7; Johnston et al. 1994 Meth Cell Biol 43:353-65), and jet injection (Furth et al. 1995 Molec Biotech 4:121-7). Expression of an exogenous DNA has also been accomplished by direct application of DNA or DNA-liposome complexes to incisional wounds (Sun et al. 1997 J Invest Dermatol 108:313-8). Semi-invasive methods involve permeabilization of the epithelium through either mechanical or chemical means. For example, one successful semi-invasive method involves the application of a pulsed electric field to the skin (Zhang et al. 1996 Biochem Biophys Res Commun 220:633-6). The invasive and semi-invasive methods generally deliver the polynucleotide in the form of naked DNA (see, e.g., U.S. Pat. No. 5,589,466). Non-invasive methods include topical application of a DNA-containing formulation that contains transfection-facilitating molecules. Examples of such formulations include liposomes (Li et al. 1995 Nature Med 1:705-6; Alexander et al. 1995 Human Mol Genet 4:2279-85). In general, conventional non-invasive methods involve pretreatment of the skin to remove hair (e.g., by shaving and/or use of a depilatory) (Li et al. 1995 Nature Med 1:705-6; Alexander et al. 1995 Human Mol Genet 4:2279-85).
Although conventional methods hold great promise for delivery of gene products to the skin of local and systemic effects, the more complicated the delivery method or the delivery formulation, the more difficult application of these methods and formulations will be in the field. For example, a genetic vaccine preferred for use in the field would be one that requires no special equipment, such as instruments for breaking the skin to deliver the DNA, and further involves no special formulation that might require special handling. Methods that use needles or require multiple dosages via an invasive route meet with problems of patient compliance. Finally, it would be desirable to have a means to avoid the use of recombinant viruses, which may have undesirable side effects and safety concerns.
There is a need in the field for methods of delivery of polynucleotides to skin cells that does not require special formulations or invasive procedures to facilitate delivery of the genetic material into skin cells. The present invention addresses this problem.